Abstract The influenza M2 protein forms a pH-activated proton channel that is essential for the virus lifecycle. Inhibition of the H+ channel activity by the amantadine class of antiviral drugs has been made ineffective by mutations in the M2 transmembrane domain. High-resolution structure determination of M2 is thus paramount for developing new antiviral drugs to target amantadine- resistant M2 variants. This small protein contains all the machinery necessary for pH activation, H+ selectivity, and gating, and is thus an excellent model system for understanding larger voltage-gated H+ channels and pH-gated ion channels. Work funded by this research proposal has previously led to the elucidation of the pharmacologically relevant drug-binding site in M2 and revealed pH-dependent dynamics of the proton-selective residue, His37. However, new H+ conduction models have since been proposed, and the structure basis for channel gating by Trp41 has not been studied. Aim 1 of this proposal is to elucidate the H+ conduction mechanism of M2 by examining His37 structure at acidic pH when the channel is activated. H-bonding, protonation/deprotonation dynamics, and the effects of inhibitors on His37 structure will be measured. Both amantadine and Cu2+ will be used as inhibitors. Aim 2 is to elucidate Trp41 structure and interaction with His37 by measuring chemical shifts and inter-atomic distances. In addition to the H+ channel activity, M2 mediates virus budding by causing membrane curvature in a cholesterol-dependent fashion. We will investigate M2-membrane and M2-cholesterol interactions by distance and relaxation NMR measurements. Aim 3 will measure whether M2 preferentially binds to highly curved regions of the membrane. M2 interacts with the matrix protein M1 through its cytoplasmic tail during virus assembly and budding. No structural information is yet available for the cytoplasmic domain. In aim 4 we will determine the 3D structure of full-length M2 in lipid bilayers using multidimensional MAS correlation experiments.